It has been known for more than 100 years that various diseases are caused by breathing airborne asbestos fibres, which are now known to include pleural and pulmonary asbestosis, lung cancer, mesothelioma, and possibly some other cancers.
Crude methods of measurement of asbestos fibres commenced around 1900, with a number of different techniques being developed over the next 60 years.
The current method was developed during the 1960's, which samples a measured volume of contaminated air through a 0.8 pore size membrane filter. The filter is then mounted onto a microscope slide, and chemicals are applied so as to make the filter transparent.
After applying a coverslip, a Phase Contrast Optical Microscope (PCOM) is used to count all fibres which are defined geometrically as being greater than 5 μm long, less than 3 μm wide, and have a length:width ratio greater than 3:1.
Mineralogical studies had previously shown that asbestos fibres are crystals that had grown in such a way as to form long fibres, and when viewed under conventional (bright field) microscopy were invisible.
Thus, a means had to be found to convert the transparent fibres into ‘amplitude’ specimens—that is, able to be seen because there is a difference in light contrast between the fibres and that of the surrounding collection filter.
Asbestos fibres have a higher refractive index than the collection filter and are known as ‘phase objects’ because the light that passes through them travels at different speeds than through the filter—thus causing a phase shift in the light wavelength that is proportional to the ‘optical path length’ difference’ (OPL Diff), which in turn is numerically equal to the diameter of the fibre multiplied by the difference in refractive indices. It is not uncommon to describe a phase object in terms of Phase Angle (in units of degrees) which is defined as the OPL Diff multiplied by 360 and divided by the wavelength of light, generally taken as 500 nm for phase contrast microscope applications; or in terms of Phase Angle (in units of mrad) which is defined as the OPL Diff multiplied by 1,000×2×π and divided by the wavelength of light, generally taken as 500 nm. The PCOM employs partial destructive interference of light, which converts fibre ‘phase objects’ into ‘amplitude objects’ because of its design.
PCOM is however a qualitative tool designed for originally observing biological matter, and if any of a number of parameters of the microscope design are changed, or if the configuration of a microscope is so adjusted or changed, this leads to changes in the microscope performance and hence to different levels of fibre detectability. For example, one microscope may be able to detect the presence of 0.05 μm diameter chrysotile asbestos fibres whereas another equally good quality microscope can only detect the presence of 0.2 μm diameter chrysotile fibres. Observer experience, conscientiousness, fatigue and eye acuity is also an important factor in the level of fibre detectability. It is only by testing the observer/microscope performance can these differences be eliminated and the concept of a ‘standardised’ microscope achieved.
Over the years, various failed attempts at producing a phase contrast detection limit test slide included the use of diatoms or microspheres, and the proposed used of insect web or quartz micro fibres.
In the late 1970's and early 1980's the United Kingdom (UK) Health and Safety Laboratory (HSL) of the UK Health and Safety Executive (HSE) and the UK National Physical Laboratory (NPL) collaborated and produced the first Phase-Contrast Test Slide (PCTS), known in this document as the ‘HSE/NPL PCTS’.
The HSE/NPL PCTS is a conventional glass microscope slide which contains ridges of resin of various depths in seven sets, No. 1 to No. 7, which are covered with a second resin of slightly lower refractive index, which in turn is covered by a glass coverslip.
In use, observers must use the HSE/NPL PCTS on a daily basis, and must be able to detect all of the ridges in the first five sets, and optionally some of the ridges in the sixth set, but never the ridges in the seventh set.
The creation of the ridges was originally accomplished by the NPL by first using a ‘ruling engine’ (normally used for creating diffraction gratings) to rule grooves with a ‘V’ shaped diamond tool in an aluminium film which had been deposited on an optically flat glass substrate. The grooves were then replicated using resin, and placed onto glass microscope slide such that the grooves were uppermost thus creating ridges. A synthetic resin known as Euparal was then deposited on the replicate, over which a glass coverslip was placed.
The depth of each of the ridges is around ten times less than the width of the ridges, which in conjunction with the Euparal forms phase objects having an approximate OPD determined by the depth of the ridges and the difference in refractive indices between the ridge material and the Euparal. See Table 1.
TABLE 1DescriptionOPL Diff (nm)Phase Angle (°)Phase Angle (mrad)HSE/NPL - No 11087.4129HSE/NPL - No 2775.393HSE/NPL - No 3644.477HSE/NPL - No 4533.663HSE/NPL - No 5443.052HSE/NPL - No 6362.544HSE/NPL - No 7251.730
The manufacture of the current UK HSE/NPL Phase Contrast Test Slide (PCTS) is very labour intensive, subject to errors in fabrication, variable in quality, has high rejection rates, and is expensive to produce.
From approximately mid 2016 to at least mid 2017, the mandatory UK Health and Safety Laboratory (HSL) inspection of every PCTS produced by a third party resulted in the rejection of a high percentage of devices because they did not perform satisfactorily.
The reasons for the inspection failures are not clear, even though it is believed to be due to manufacturing problems which have been difficult to overcome with any stability or certainty.
These failures have led to major problems for a number of laboratories throughout the world because the use of the HSE/NPL PCTS is mandated by various Country statutory and accreditation authorities, and also is required by the various analytical methods required to perform airborne asbestos fibre analyses. It is reported that some of the HSE PCTS have been stolen from various laboratories for use in other laboratories.
In essence, the current PCTS pushes the very limit of 1960's technology which would be impossible to maintain without considerable effort and expense.
The above mentioned old technology limited the presence of phase object locating ‘guides’ to rudimentary parallel and horizontal lines that are not very efficient effective in assisting the user in locating near invisible sets of phase objects. This deficiency is exacerbated by an extremely small microscope depth of field, and a stage which holds the test slide not being sufficiently stable to keep the phase object in focus when moving the slide from one set of phase object to another. Thus the phase objects often go out of focus thus becoming completely invisible.
The above mentioned old technology has prevented the use of sufficient space between, or identifying marks for any of the seven sets of phase objects, which leads to the user often having to backtrack over previously observed sets to make sure which particular set of phase objects are being observed. These deficiencies lead to frustration, loss of time, and sometimes incorrect results.
Accordingly there exists a need for an improved phase contrast detection limit test slide that seeks to overcome or at least ameliorate some of the problems of the prior art.